Selenium deficiency is associated with the development of a cardiomyopathy in children and has been postulated to play a role in diseases such as cancer, cirrhosis, and myocardial infarction. Selenium functions biochemically as a constituent of selenoenzymes. Recently a rat plasma selenoprotein has been purified and characterized. It contains over 60% of the plasma selenium and has 10 selenocysteines per molecule (deduced from cDNA). This protein has been called selenoprotein P (Se-P) and proof has been presented that a similar selenoprotein exists in human plasma. Preliminary studies presented in this proposal demonstrate the production of monoclonal antibodies to human Se-P. 75Se-labeled material secreted by HepG2 cells was mixed with human plasma and a partial purification was carried out using 75Se as the marker. This material was used as an antigen source. Two monoclonal antibodies were produced and have been used to make immunoaffinity columns and purify Se-P from human plasma. The purified material yields 2 bands on SDS-PAGE unlike rat Se-P which yields 1. Both bands contain carbohydrate. A cDNA clone has been isolated from a human liver cDNA library and sequenced. It has 74% identity of deduced amino acid sequence with the rat Se-P. This proposal would separate the 2 forms and characterize them with respect to selenium and amino acid content. Peptide analyses would be carried out to determine if the smaller one is a shorter version of the larger one. Other possibilities would also be considered. Regulation of synthesis of Se-P and of cellular and plasma glutathione peroxidase would be studied using HepG2 cells as a model. These cells can be made selenium deficient and mRNA and protein levels of the selenoproteins would be measured. Molecular genetic studies including construction of cDNA libraries from selenium-deficient and control HepG2 cells with cloning and sequencing of cDNAs of Se-P from both would also be done to determine whether selenium deficiency affects Se-P mRNA. A radioimmunoassay for human Se-P would be developed and used to establish a normal concentration range. Then a study would be carried out to measure Se-P in patients with liver disease. The assay would be used to measure Se-P in plasma from subjects consuming different amounts of selenium. These studies should provide insight into selenium metabolism and function in human beings and move us closer to understanding the relationship of selenium to human disease. They should also reveal how Se-P relates to glutathione peroxidase activity and whether measuring Se-P in plasma is useful as an assessment of selenium status.